It has recently been discovered that cloned genes can be efficiently and stably introduced into the mouse germ line by microinjection into the fertilized egg. Combined with current techniques for gene isolation and for the analysis of gene expression in the organism, this promises to provide a powerful approach to the study of gene regulation during mammalian development. In the proposed studies, we will microinject several cloned Beta-globin genes, along with varying amounts of flanking genomic DNA, into mouse eggs and derive lines of mice carrying these genes in the germ line. Using molecular biological techniques, we will examine the expression of the exogenous globin genes in erythroid and non-erythroid mouse tissues. This approach should allow us to locate regulatory sequences involved in the erythroid cell-specific and stage-specific activation of the globin genes. We will also examine the de novo methylation of exogenous DNA sequences introduced into the genomes of developing mice, and the possiblity that these sequences may undergo subsequent de-methylation events in certain cell lineages. The methylation patterns of exogenous genes should reflect the normal mechanisms by which tissue-specific DNA methylation patterns are established and altered during development, and methylation may also be an important factor in determining the capacity of exogenous genes for proper expression in the animal. In another genes of experiments, we will investigate the process by which cloned genes are integrated into a host chromosome after injection into the mouse egg, and the consequent structure of the integrated genes. Focusing on the globin genes, thes studies will begin to explore the capabilities of gene transfer into the mouse as a general method for the investigation of developmental gene regulation. They are thus directly relavent to our understanding of genetic defects of hemoglobin synthesis, and their basic relevance may extend to other diseases involving abnormal gene expression. Information concerning the structure and expression of exogenous genes in the cells of whole organisms will also be useful in assessing the eventual feasibility of using gene gransfer to treat human genetic diseases.